ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is the world?s 6th leading cause of cancer, accounting for 5% of cancer mortality. For many other cancers, molecular characterization has led to sub-classification of disease based on the status of oncogenic drivers in signal transduction pathways, leading to the development of molecularly-targeted agents (MTAs), targeting specific dysregulated proteins in these pathways. HNSCC genetics are highly complex with no clinically actionable mutations or classification beyond HPV-positive and HPV-negative tumors. Classification to improve therapeutic decision-making is needed for HNSCC. Cetuximab (CTX), an epidermal growth factor receptor (EGFR)-binding monoclonal antibody (mAb), is FDA- approved for HNSCC as a monotherapy or combined with radiation or platinum-based therapy. Though having a substantial clinical impact in a small proportion (~13%) of patients, treatment with CTX is associated with serious adverse reactions, resulting in interrupted therapy in 3-10% of patients. Due to this high risk, along with a high cost and the low response rate, a companion diagnostic (CDx) to identify patients most likely to respond to CTX is urgently required. We propose to develop a live tumor cell based CDx to guide the identification of CTX-responsive patients based on tumor ex vivo functional responses which are predictive of clinical outcomes. Since traditional tissue processing strips tumor cells of their biological integrity it is only suitable for histologic review, not allowing analysis of dynamic biomarkers. BioMarker Strategies is developing the SnapPath Process and Pathology Multi Analyte Profiles (PathMAP) technologies for live tissue processing of solid tumors using a single-use cartridge, which holds the sample and all the necessary reagents and consumables, automating and standardizing tumor processing and treatment to enable highly predictive and standardized tests. The tumor cells' responses are scored against PathMAP, a locked classification model. PathMAP tests can help guide medical oncologists in their treatment decisions for cancer patients. The proposed CDx will measure the pharmacodynamic response upon exposure to CTX of specific phosphoproteins in MAPK, JAK/STAT, and PI3K pathways, the key pathways in HNSCC pathophysiology. We propose to: 1) Optimize Conditions and Response Profile to Predict CTX Sensitivity to detect strong pharmacodynamic responses in sensitive cells and minimal responses in resistant cells; 2) Evoke Functional Signaling Profiles (FSP) Against CTX in Complex Samples by first using clinically- relevant patient derived xenograft (PDX) samples and then human clinical samples. The proposed research will serve as a feasibility study determining percent suppression cut-off values for predictive biomarkers and confirming operationalization of the test in complex samples. It will also determine the sample size for a future larger multisite sample collection in Phase II.